In the present study distinct spatial differences in stable isotope fractionation patterns were observed for C. gariepinus and parasites from the Vaal River. In host fish, highest δ15N levels were recorded from samples collected below the Vaal River Barrage, whereas, samples collected from the site below Grootdraai Dam showed lowest stable nitrogen isotope levels. The opposite was observed for 13C enrichment in hosts among the different sites, with lowest levels observed in C. gariepinus collected below the Vaal River Barrage. These differences may be related to the variability in the diet of C. gariepinus as a result of availability of prey items at each sampling site along the Vaal River. The Sharptooth catfish is a typical omnivore, feeding on a wide variety of organic matter with prey items varying from birds, reptiles, other fish species, including smaller C. gariepinus, to macroinvertebrates and plant material [37]. As stable carbon isotope ratios serve as a representation of the nutrient sources in food webs [38], comparison of differences in the enrichment of 13C isotope can be considered a good indicator of spatial variation in baseline isotope levels [34, 39]. The variability of δ13C in muscle tissue of C. gariepinus between sampling sites suggests that a wide range of dietary items are consumed by this fish species. The present results further support a dietary shift in C. gariepinus inhabiting the Vaal River. Negative correlation between host morphometry and δ13C indicate that the foraging habits and food sources utilised change as fish grow. This was further supported by a positive correlation between host length and weight with δ15N values. In a previous study, Kadye and Booth [40] showed that although there is an apparent dietary shift in C. gariepinus with fish growth, there is a high dietary overlap between fish of different size classes which can be related to the omnivorous feeding strategy of this fish species. In the present study collections were performed in spring which coincided with the period when C. gariepinus prepare to begin spawning in the summer following rains [37]. Differences in condition factor (K) between male and female host fish (Kfemale fish > Kmale fish) can therefore be related to the larger weight of gravid female fish. Spatially, there was no difference in the condition factor of the hosts between the different sampling points along the Vaal River and therefore with the levels of stable nitrogen and carbon isotopes. Clarias gariepinus serves as a host for a wide variety of endoparasite and ectoparasite taxa [41] and this could likely result from the variable diet and wide distribution of this fish species. In the present study the association between parasite intensity and host morphometry was variable. Comparisons with P. glanduligerus were not performed due to the low number of samples obtained. Positive correlation between the size and weight of C. gariepinus hosts and L. clariae, T. ciliotheca and larval Contracaecum sp. indicate that larger fish harbour more parasites than smaller ones. This was especially so for T. ciliotheca infecting C. gariepinus which showed a significant and positive correlation with host morphometry. The concept of larger fish harbouring higher parasite intensity has been well documented in previous studies (see [42]).
Isotopic discrimination of 15N indicated that all nematode larvae and cestodes collected from C. gariepinus were depleted relative to the host fish from all sites. Depletion of the heavier stable nitrogen isotope in cestodes and larval nematodes is in line with previous findings for other cestodes and larval nematodes [20, 29] infecting fish hosts, and for some cestodes infecting rabbit hosts [19]. Differences in δ15N between larval nematodes and cestodes with host muscle tissue accounted for shifts of approximately one to two trophic levels, respectively. These observed differences are in the range for other host – cestode and larval nematode systems analysed [19–21, 23, 24, 26] and can be related to the mode of nutrient acquisition. For cestodes, lack of a digestive system has meant that the tegument of these organisms has become specially modified for the accumulation of molecules derived from the hosts’ metabolism [43]. As a result of transamination of complex proteins, the heavier stable nitrogen isotope is retained in the tissues of the host [44] and as a result endoparasites are depleted in heavier isotopes. Comparison of stable nitrogen isotope levels between endoparasites showed that larval Contracaecum sp. has the least depletion. This can be related to the fact that the larval stages of these nematodes are not actively feeding on host tissue or metabolic compounds as adult cestodes do.
Results for isotopic discrimination between host muscle tissue and Contracaecum sp. larvae in the present study are in line with previous studies for other larval nematodes encysted in the peritoneal cavity of host fish [8, 18]. In the case of larvae of Contracaecum sp. infecting C. gariepinus, nematodes were encysted in the mesenteries of the viscera in the region of the intestinal tract. According to Moravec et al. [45] these larval stages exhibit low pathogenicity and along with the current 13C and 15N isotope data indicate that the nematodes are not actively feeding on fish hosts. Rather Moravec et al. [45] suggests that these larval nematodes develop to the third stage in the egg in the water column which is then ingested by a fish which functions as a paratenic host. Thus, it is plausible that the lack of a relationship between host stable isotopes and larval nematodes is the result of C. gariepinus being a paratenic host to Contracaecum sp. larvae which are not actively feeding on the host. Additionally, Nachev et al. [8] indicated that the slow growth rate of some nematode larvae is a contributing factor resulting in the lack of a relationship between host stable isotopes and larval nematodes. In order to determine if these larval stages derive some nutrition from paratenic hosts, comparisons of stable isotopes for different larval stages will have to be performed. In South Africa, studies have indicated that larval Contracaecum sp. are widespread and occur frequently in C. gariepinus [see 39, 40]. Despite this, 15N fractionation observed in the present study indicated that endoparasite taxa infecting C. gariepinus occupy relatively similar trophic levels. Similarly δ13C for all endoparasite taxa fall within the 1–2‰ range of enrichment factors reported in previous studies [21, 38, 47]. For cestodes and encysted larval nematodes, differences in carbon stable isotope signatures are comparable to other reports for similar host – parasite systems [18, 21, 25] and indicate that nutrients assimilated by the host and parasites are from similar sources [21].
Regarding the copepod, L. clariae, fraction of both 15N and 13C showed that the parasites were variably enriched and depleted in both stable isotopes compared with host muscle tissue. Based on differences in the heavier nitrogen isotope this did not correspond to differences in trophic position and therefore host and female copepods likely occupy similar trophic positions. Instances where adult female L. clariae were 15N enriched relative to host muscle tissue correlates with the feeding pattern of the parasite and corroborates stable 15N isotope fractionation patterns in other haemophagous ectoparasites [9, 19, 32, 48, 49]. The variability in 15N fractionation between C. gariepinus and L. clariae observed in the present study further corroborates findings for other parasitic copepods, which parasitise the gills of the host fishes and share similar feeding biology as L. clariae [18, 20, 29]. Iken et al. [29] found that a copepod infecting gills of Coryphaenoides armatus was enriched by 2.7‰ in 15N relative to the host. Pinnegar et al. [20] found that Lernaeocera branchialis, infecting the gills of the host fish, Platichthys flesus, were depleted in 15N stable isotope by 0.81‰ relative to the host. Unlike L. clariae which feeds on blood from the gill filaments, L. branchialis feeds on blood from the bulbous arteriosus of the heart of infected fish hosts, where it macerates host tissue with its mandibles and feeds on host blood [see 45]. In an assessment of isotope fractionation between several copepods infecting the gills of their hosts, Deudero et al. [18] showed high interspecific variation in 15N enrichment between copepod species feeding on the same host fish. For instance, they found that Clavella adunca feeding on both cod and whiting were depleted in 15N stable isotope but L. branchialis feeding on whiting or haddock were enriched in 15N but were depleted in 15N when parasitising cod. Similarly with L. clariae, the copepods analysed by Pinnegar et al. [20] and Deudero et al. [18] infected the gills and feed on the blood of the host fishes. However, Shotter [51] indicated that the mandibles of Clavella uncinata, a similar species to C. adunca, were too weak to tear tissue but instead function in gathering material by scraping superficial tissue toward the mouth, with little blood comprising the diet and intestinal contents. In some instances, studies have indicated adult female copepods are significantly enriched in 15N relative to the host organism [52–54]. Gretsy and Qyarmby [52] found that adult Mytilicola intestinalis were enriched by 3‰ relative to the intestine of European blue mussel host (Mytilus edulis). Goedknegt et al. [54] similarly found that adult Mytilicola orientalis were enriched in 15N stable isotope relative to the adductor muscle of the host mussel, M. edulis, by 1.2‰. In both instances the higher 15N enrichment of both species could be related to the parasites feeding directly on the intestinal tissue of the host. In a seasonal study on the gut ultrastructure and contents in conjunction with stable carbon and nitrogen isotope analysis of Neoergasilus japonicus, Baud et al. [53] showed that adult female parasites infecting the fins of the host fish, Perca fluviatilis, were enriched in the 15N stable isotope by 3.7‰ relative to the host muscle tissue, indicating that the parasite feeds on host tissue. The high variability in 15N enrichment of parasitic copepods may be related to the variability in stable isotope enrichment of host tissues consumed by parasites as well as the isotopic enrichment of the host resulting from dietary variation in cases were parasitic copepods feed on similar host tissues. In the present study, spatially variable 15N fractionation patterns of L. clariae infecting the same host corresponded or mirrored spatial 15N isotope enrichment of the host among the collection sites along the Vaal River. In most instances, adult L. clariae were slightly enriched in 15N stable isotope relative to the host fish tissues (VRGD, VD and VRHD), but were also slightly depleted relative to the host fish at sites below the Vaal River Barrage and at Bloemhof Dam. Adult L. clariae and egg strings shared similar stable isotope signatures. High standard deviations in the stable isotope levels of L. clariae could be related to spatial differences in the food sources consumed by the host fish. Additionally, during their life cycles many parasitic Copepoda undergo drastic morphological changes from free swimming larval stages which are able to move between hosts to sedentary parasitic adults. It is possible that during the free swimming larval stages, copepodite stages may feed on hosts of variable isotope enrichment as well as incorporate other sources of nutrition before becoming parasitic on a host fish. The variation in the diet during the free swimming larval stages may then further result in variations in stable isotope enrichment observed in adult organisms.
According to predictions of stable isotope fractionation between consumers and prey items, geographic variation in the ratios of stable isotopes of carbon and nitrogen should be reflected in a predictable manner in relation to the isotope signatures of the host [6, 47, 55]. As such variability in the diet of the host should be reflected more prominently in the stable isotope composition of endoparasites more so than ectoparasites [18]. However, from the results of the present study, variations in the host diet were more closely mirrored in the isotope enrichment of L. clariae than endoparasites. Iken et al. [29] observed no similarity in isotope enrichment in trematodes feeding on intestinal tissue of the host fishes, Chalinura profundicola and Chalinura leptolepis, while a copepod feeding on a gastropod host, Oneirphanta mutabilis, did exhibit similarity in isotope fractionation. Deudero et al. [18] similarly noted that stomach nematodes did not reflect isotopic differences observed in host fishes. Sures et al. [9] showed that in a monogenean – host fish system from the Vaal Dam, the micropredatory nature of P. ichthyoxanthon resulted in mirroring in isotope fractionation between two yellowfish hosts.
Spatial geographic differences observed at various trophic levels can be related to host – specific differences in ecology and behaviour [18, 34]. Regarding the spatial differences observed in isotopic enrichment patterns of parasite taxa in the present study, as the feeding strategies of the parasites do not change between the different sites, the differences in enrichment patterns observed are likely related to differences in food items utilised by the host fish. The spatial variability in stable isotopes in parasites infecting C. gariepinus therefore reflects spatial differences in the baseline isotope signatures across the distribution of the host. Geographical differences in δ13C and δ15N levels of organisms has previously been documented [39]. With regard to parasites, Gómez-Díaz and González-Solís [34] similarly observed spatial differences in the isotopic signatures of ectoparasites infecting two closely related shearwater hosts, Calonectris diomedea and Calonectris borealis, across the Mediterranean Sea and Northeast Atlantic. Low variation in stable isotope composition of parasites collected from the Vaal River can be related to the fact that parasites do not change the resources they utilise from the host and rather spatial differences in parasite stable isotope enrichment is related to the variance in host diet. This was similarly observed by Riascos et al. [56] for Hyperia curticephala infecting the scyphomedusa, Chrysaora plocamia. It should also be noted that unevenness of the prevalence and abundance of parasites along the Vaal River likely lead to a lop-sided sampling design and as such spatial differences in parasite stable isotope enrichment observed in the present study should be confirmed following more even sample collection.
Comparison between the parasites infecting C. gariepinus showed that L. clariae were significantly and constantly enriched in 15N stable isotope compared to the endoparasites. The shift in δ15N observed for endoparasites and L. clariae overall accounted for a difference of approximately two trophic levels. Variability in the signatures reported between the different parasite taxa analysed are similar to other studies which have found similar co-infections [18, 20, 29]. The differences in δ15N can be linked with differences in nutrient acquisition strategies by each parasite taxon. Mature, adult female L. clariae consume whole blood and epithelial cells which they acquire from the secondary gill filaments of the host fish [57, 58]. Mature female copepods attach to the gill filaments using their maxillipeds and feed on gill epithelium and blood using the maxillae which scrape cellular material toward the mouth and along with a negative pressure created by the muscular oesophagus, blood and cellular debris are sucked into the buccal cavity [57–59]. Unlike helminth endoparasites, some copepod ectoparasites are not able to accumulate simple amino acids across the keratinised body surface and instead must break down complex proteins which are accumulated in meals [18]. As a result these parasites are generally enriched in heavier 15N stable isotopes in a manner similar to consumer organisms which resembles a predator–prey relationship. Whereas, in the case of both cestodes and larval nematodes, feeding can be likened to that of a commensalistic scavenger, whereby, the parasites feed on left over by-products of the hosts metabolism and in doing so pose little harm during feeding toward the host.